ALBERT

Description: Background. T-cell depletion is often used to reduce graft-vs-host disease in haploidentical donor hematopoietic cell transplantation (HCT). However, the absence of adoptively transferred T-cells may increase graft failure, relapse, and infection. Novel methods have been developed to more selectively deplete naïve T cells and preserve memory T cells. Depletion of CD45RA+ cells can provide robust early recovery of diverse memory T-cell populations in haploidentical donor transplantation, and may provide increased immunity against viral infections. Likewise, the provision of additional donor NK cells may reduce viral complications. Patients and Methods. Sixty-seven patients received initial allogeneic HCT on 3 consecutive IRB approved haploidentical donor HCT trials at St. Jude Children's Research Hospital from 2005 to 2015. CD3-depletion was used in 41 recipients, and CD45RA-depletion was used in 26. All patients received similar preparative doses of fludarabine, thiotepa, and melphalan. Patients with CD3-depleted grafts received OKT3 (n=20) or Campath (n=21), and all 41 received rituximab on Day 0 as EBV prophylaxis. Patients with CD45RA-depleted grafts (n=26) did not receive antibody therapy, but instead received total lymphoid irradiation and a dose of cyclophosphamide added to the preparative backbone. Donor NK cells were given Day +6. Peripheral blood was tested for CMV, EBV, and adenovirus using quantitative PCR at least weekly until day +100, and then as indicated. The first 180 days post-HCT were evaluated. Fisher's exact test was used to compare two proportions. Results. Patients with CD3-depletion received a median 0.04 (range: 0.01 - 0.15) x 106 CD3+ cells/kg, and patients with CD45RA-depletion received a median 80.07 (range: 16.08 - 528.52) x 106 CD3+ cells/kg. CMV reactivation occurred in 23 of 41 patients (56.1%) with CD3-depletion and 5 of 26 patients (19.2%) with CD45RA-depletion (p=0.005). Differences occurred predominantly in those CMV seropositive recipients who received grafts from CMV seropositive donors, as CMV was detected in 22 of 24 (91.7%) +/+ patients with CD3-depletion and 4 of 11 (36.4%) +/+ patients with CD45RA-depletion (p=0.001). Of the 23 patients with CMV after CD3-depletion, the peak viral load was a median 4.49 log10 copies/mL blood (range:

Description: The two lineages of T cells, αβ and γδ, diverge early in development and recognize ligands differently. γδ T cells are not restricted by the major histocompatibility complex (MHC)and can recognize peptides, non-peptides as well as small molecules. The effector functions of αβ and γδ T cells are also biologically distinct. Mature T cell lymphomas derived from the γδ lineage are often aggressive with poor prognosis. For T cell acute lymphoblastic leukemia (T-ALL), it is customary to view the TCR expression on lymphoblasts as an indicator for the maturational state, although identification of the heterodimer can distinguish two distinct T cells. The αβ and γδ T cells are derived from different lineages with distinct developmental stages and biological characteristics. Here we evaluated the clinical presentation and outcomes of patients with γδ T-ALL who were treated on protocol to determine the impact of lineage specification on T-ALL prognosis. From January 2004 to July 2014, 100 consecutive patients with newly diagnosed T- ALL were enrolled onto the Total Therapy XV or XVI study at St. Jude Children's Research Hospital (SJCRH). Of the 100 patients, 93 had diagnostic samples screened and confirmed for γδ TCR and were evaluable for follow up. Patients received risk-adaptive treatment consisted of remission induction, consolidation and continuation as previously described. Of the 93 patients, 12 (13%) had γδ T-ALL. With the exception of higher percentage of γδT-ALL patients classified to have high risk disease based on high MRD levels (≥1%) at the end of remission induction (25% vs. 11%; P =0.04), there was no significant differences between the two groups in regards to age, gender, race, presenting leukocyte count, presence of a mediastinal mass and CNS involvement at diagnosis. Patients with γδ T-ALL had a significantly higher risk for induction failure with MRD ≥ 1% at day 15 (67% vs. 33%; P =0.006) and day 42 (25% vs. 11%; P =0.04). High portion of patients with γδ T-ALL (40%) were referred for an allogeneic hematopoietic stem cell transplantation. The overall survival was significantly lower for patients with γδ T-ALL compared to non-γδT-ALL at 5 years (63 ± 20% vs. 91 ± 4%) and 10 years (63 ± 38% vs. 88 ± 31%) (P =0.01). The event free survival was lower and relapse rate was higher for the γδ T-ALL patients compared to the non- γδ T-ALL patients, although not significant. Majority of patients with γδ T-ALL had complex cytogenetic abnormalities (91%) and one patient had normal karyotype. Interestingly, hyperdiploidy (47-53) was common, but only one patient had high hyperdiploidy with a DNA index of 1.17. Immunophenotype showed variable expression of CD34, CD1a, CD2 and CD7. The pairing of the Vγ and Vδ chains expressed by the lymphoblast was identified using high throughput single cell PCR on the expressed cDNA. Out of 12 patients, 9 patients had appropriate consents, samples and successful amplification The TCR was predominately Vδ1 (67%) and the CDRγ region was highly conserved (~88%) between patients. Despite the broad diversity of the rearranged TRG region, the CDR3γ region was highly conserved. The CDR3γ region contained an average of 14.2±2.2 amino acids and each patient had an average of 3.3 ± 2.6 unique amino acids. An average of 11 ± 0.5 amino acids (88%) were conserved amongst the T-ALL patients. All the patients used the terminal TRGC2, suggesting a bias towards a non-disulfide linked TCR. Majority (〉75%) of γδ T cell in normal peripheral blood is Vγ9Vδ2, and none of the patients expressed Vδ2. The dominant Vδ1 has been shown to be resistant to activation induced cellular death (AICD) and can persist for many years. This may correlate to the recent association of SET-NUP214, a fusion gene involving 9q34 that is specific for the γδ lineage and confers chemotherapy resistance. Further studies evaluating the genetic alternations will shed insight into to potential mechanisms. In summary, expression of the γδ TCR on T-ALL lymphoblasts identified a subset of patients with high risk for induction failure and poor outcome. Disclosures Evans: Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping.

Description: Notch signaling regulates multiple cell fate decisions by hematopoietic precursors. Previously, we found that endogenous Notch signaling in cultures of murine hematopoietic precursors (Lin-Sca-1+ c-Kit+) leads to a multi-log increase in the number of Sca-1+ c-Kit+ cells, inhibition of myeloid differentiation, and promotion of T/NK differentiation. To activate Notch signaling in those studies, a single dose (10μg/ml) of engineered Notch ligand consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG1 (Delta1ext-IgG) was immobilized to the plastic tissue culture surface. To investigate quantitative effects of Notch signaling, bone marrow Lin-Sca-1+ c-Kit+ (LSK) cells were cultured with plates coated with increasing concentrations of Delta1ext-IgG in media supplemented with 20% FBS, SCF (100 ng/mL), Flt3L (100 ng/mL), IL6 (100ng/mL) and IL11 (10ng/mL). LSK cells cultured for 14 days with control human IgG1 underwent terminal myeloid differentiation (determined by expression of GR1 and F4/80) with no further increase in cell number, whereas at all densities of Delta1ext-IgG there was approximately a 3 log greater number of cells than in control cultures. Furthermore, the portion of cells that maintained Sca-1 and c-Kit expression increased at greater densities of Delta1ext-IgG (10%, 32%, 77%, 71%, 71% and 71% for plates coated with ligand at 0.6, 1.25, 2.5, 5, 10 and 20 μg/ml, respectively, and 5% for human IgG1 control at 10μg/ml), whereas the portion of cells undergoing myeloid differentiation decreased at greater ligand densities (48%, 33%, 5%, 3%, 3% and 3% respectively, and 40% for control). In contrast, a substantial increase in the portion of cells expressing B220+ was observed at relatively low densities of Delta1ext-IgG (30% at 0.6 μg/ml and 19% at 1.25 μg/ml) compared to control (4%), but was no longer evident with further increases in ligand density (1.8%, 2%, 1.2%, m1.6% at 2.5, 5, 10 and 20 μg/ml respectively). Furthermore, promotion of early T cell differentiation was observed in ligand containing cultures with the generation of increased number of cells co-expressing Thy1.2 and CD25 (14%, 24%, 22% and 24% at 2.5, 5, 10 and 20 μg/ml respectively). Further evidence for T cell commitment was established by quantitative RT-PCR in which increased expression of CD3ε and pre-Tα was observed by 28 days of culture. Thus these studies demonstrate that culture with different densities of the Notch ligand, Delta1ext-IgG results in differential cell fate outcome with inhibition of myeloid differentiation and promotion of early T cell induction that is maximal at high ligand densities and of B220+ cells at relatively lower densities.

Description: In patients with acute leukemia, detection of minimal residual disease (MRD) before allogeneic hematopoietic cell transplantation (HCT) correlates with risk of relapse. However, the level of MRD that is most likely to preclude cure by HCT is unclear, and the benefit of further chemotherapy to reduce MRD before HCT is unknown. In 122 children with very-high-risk acute lymphoblastic leukemia (ALL; n = 64) or acute myeloid leukemia (AML, n = 58), higher MRD levels at the time of HCT predicted a poorer survival after HCT (P = .0019); MRD was an independent prognostic factor in a multivariate analysis (P = .0035). However, the increase in risk of death associated with a similar increment of MRD was greater in ALL than in AML, suggesting that a pretransplantation reduction of leukemia burden would have a higher impact in ALL. At any given MRD level, survival rates were higher for patients treated in recent protocols: the 5-year overall survival for patients with ALL was 49% if MRD was detectable and 88% if it was not and the corresponding rates for patients with AML were 67% and 80%, respectively. Although MRD before HCT is a strong prognostic factor, its impact has diminished and should not be regarded as a contraindication for HCT.

Description: We evaluated 190 children with very high-risk leukemia, who underwent allogeneic hematopoietic cell transplantation in 2 sequential treatment eras, to determine whether those treated with contemporary protocols had a high risk of relapse or toxic death, and whether non–HLA-identical transplantations yielded poor outcomes. For the recent cohorts, the 5-year overall survival rates were 65% for the 37 patients with acute lymphoblastic leukemia and 74% for the 46 with acute myeloid leukemia; these rates compared favorably with those of earlier cohorts (28%, n = 57; and 34%, n = 50, respectively). Improvement in the recent cohorts was observed regardless of donor type (sibling, 70% vs 24%; unrelated, 61% vs 37%; and haploidentical, 88% vs 19%), attributable to less infection (hazard ratio [HR] = 0.12; P = .005), regimen-related toxicity (HR = 0.25; P = .002), and leukemia-related death (HR = 0.40; P = .01). Survival probability was dependent on leukemia status (first remission vs more advanced disease; HR = 0.63; P = .03) or minimal residual disease (positive vs negative; HR = 2.10; P = .01) at the time of transplantation. We concluded that transplantation has improved over time and should be considered for all children with very high-risk leukemia, regardless of matched donor availability.

Description: Delayed hematopoietic recovery following cord blood transplantation (CBT) is thought to result from inadequate numbers of progenitor cells in the graft and is associated with increased early transplant related morbidity and mortality. Using an engineered form of the Notch ligand, Delta1, we have previously reported on novel ex vivo expansion methods for generating greatly increased numbers of human CD34 progenitor cells that repopulate immunodeficient mice with markedly enhanced rate and magnitude. We now report results of the initial 6 patients enrolled in a phase I study evaluating the safety and potential efficacy of cord blood (CB) progenitors cultured in the presence Delta1 and recombinant cytokines, with the goal of generating increased numbers of short term repopulating cells capable of providing rapid myeloid engraftment. These patients (AML, n=5; bi-phenotypic leukemia, n=1), were treated with a myeloablative preparative regimen consists of cytoxan 120mg/kg, fludarabine 75mg/m2 and 1320 cGy TBI, followed one day later by infusion of one non-cultured CB unit and then a second unit that has been CD34 enriched and cultured for 16 days as previously described. The median age and weight of the patients enrolled is 28 years (range 11 to 43) and 61.5 kilograms (range 26 to 76). CB units were selected on the basis of cell dose and a requirement of matching at least 4 of 6 loci with the patient (intermediate resolution for HLA-A and B, and high resolution for HLA-DRB1). The non-cultured unit in all patients was 4/6 matched to the patient. The unit used for expansion was 5/6 matched to the patient in two cases, and 4/6 matched in the other four. After culture, there was an average CD34 fold increase of 160 (range 41 to 382) with an average total nucleated cell (TNC) fold increase of 660 (range 146 to 1496). Average infused TNC/kg x107 was 2.9 (range 1.9–5.8) and 4.6 (range 0.6–9.1) for the non-cultured and cultured cells respectively, and infused CD34 cells/kg (x105) was 2.2 (range 1.1–3.4) and 53.4 (range 9.3–133) respectively. No T cells were generated during culture and no toxicities directly attributable to the cultured product, including infusional or increased acute GVHD, have been observed. All patients have engrafted. Relatively rapid engraftment was observed in 5 out of 6 patients treated to date, with a median time to engraftment of 14 days (range 7 to 34), as compared to 25 days (range 16 to 48) in patients (n=17) undergoing an identical transplant regimen at this center, but with 2 non-cultured CB units. The relative contribution of the expanded and non-cultured grafts over time was determined by a DNA-based assay for short tandem repeat loci on peripheral blood sorted cell fractions to include CD3+, CD33+, CD56+, CD14+ and CD19+ cells, beginning day +7 post transplant. In the 5 patients with early myeloid engraftment (≤20 days), engrafted myeloid cells present at day 7 were derived almost entirely from the expanded unit. In three of these five, ANC 〉500 was observed at days 7, 9 and 16 and was mainly derived from the expanded unit, whereas in the other 2 patients who achieved ANC〉500 at day 13 and 20, myeloid engraftment at day 14 was derived from the non-cultured cells. Persistent contribution to engraftment from the expanded cells has been noted in two patients, one through 280 days post transplant but no longer present at one year, and in a more recently treated patient who is currently 75 days post transplant, the expanded cells continue to dominate in CD33, CD14 and CD56, but not CD3, sorted cell fractions. Furthermore, time to platelet engraftment (〉20k) has averaged 30 days (range 19–53). Average follow-up time is 277 days (range 70–632). One patient died on day 462 from complications of VZV myelitis; all other patients are alive and in remission. Overall, accelerated myeloid engraftment has been observed in 5/6 patients treated to date as a direct result of Notch-mediated ex vivo expansion of one of two CB units prior to transplantation with ultimate engraftment from the T replete non-cultured unit. These results further suggest that improvement in early myeloid reconstitution may result from provision of short term repopulating cells and/or of cells able to facilitate engraftment of the non-cultured unit. These studies continue with the goal of achieving consistent, rapid engraftment in recipients of hematopoietic cell transplants to decrease morbidity and mortality in the early post-transplant setting.

Description: Abstract 1905 Dendritic cells (DCs) are the first committed cells to engraft in the thymus after hematopoietic stem cell transplantation (HSCT). The critical role of thymic DCs in ensuring efficient tolerance and selection has been well demonstrated, however its role in facilitating donor engraftment has not been reported. Here we show DCs accelerates thymic reconstitution by inducing regulatory T cells (Tregs) differentiation and enhancing T cell recovery after HSCT. Lethally irradiated CD45.2 C57BL/6 control group received 103 CD45.1 lin−sca-1+c-kit+ (LSK) hematopoietic stem cell progenitors while the DCs group received 103 CD45.1 LSK cells along with 103 CD45.2 GFP+ DCs. DCs were generated ex vivo using bone marrow from CD45.2 GFP+ CD57BL/6 mice and cultured for 7 days with GMCSF. At 4 and 7 days after HSCT, the thymus of DC group contained 1.8 and 4.2- fold higher number of thymocytes (p

Description: Previously, we demonstrated that activation of Notch receptors by culture of murine lin− Sca-1+c-kit+ (LSK) hematopoietic progenitors with the Notch ligand Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of LSK cells cultured with Delta1ext-IgG compared to recipients of non-cultured LSK cells or control human IgG-cultured LSK cells. Furthermore, data suggest that addition of Delta1ext-IgG cultured LSK cells to non-cultured LSK cells have the potential to accelerated T cell recovery by the non-cultured LSK cells by facilitating thymic engraftment by the non-cultured LSK cells. 103 Ly5a LSK cells cultured with Delta1ext-IgG for 21 days generated a 108-fold increase in total number of cells compared to culture with control IgG. Lethally irradiated C57Bl/6 mice received 105 C57Bl/6 GFP+ LSK cells alone or along with 103 Ly5a LSK cells or 106 Lya5a Delta1ext-IgG-cultured cells or 106 Ly5a IgG cultured cells. We monitored egraftment by difference in CD45 as well as GFP. Mice were sacraficed and blood, bone marrow and thymus were harvested at 1,2,3 and 4 wks after HCT. At 3 wks after HCT, blood samples from recipients of Delta1ext-IgG-cultured cells contained at least 4-fold higher total number of CD3+ cells compared to the other control groups (p

Description: Previously, we demonstrated that activation of Notch receptors by culture of CD34+CD38− cord blood (CB) hematopoietic progenitors with the Notch ligand Delta1ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of CB cells cultured with Delta1ext-IgG compared to recipients of control IgG-cultured or non-cultured CB cells. Furthermore, data suggest that addition of Delta1ext-IgG-cultured CB cells to a non-cultured CB graft facilitated engraftment of the non-cultured CB cells. CD34+CD38− CB cells cultured on immobilized Delta1 in serum free media with Il3, Il6, Flt3l, SCF and TPO for 16 days generated a107-fold increase in total number of cells as well as a 105-fold increase in the number of T-lymphoid biased (CD34+CD7+CD45RA+) cells. Sub-lethally irradiated (275cGy) NOD/SCIDγc−/− mice received 103 non-cultured CB cells or the progeny of 103 Delta1ext-IgG-cultured or IgG-cultured cells. At 6 and 8 wks after HCT, blood samples from recipients of Delta1ext-IgG-cultured cells contained 9- and 3-fold higher numbers of CD3+ cells compared to recipients of non-cultured CB cells (p

Description: Previously, we demonstrated that activation of Notch receptors by culture of murine lin−Sca-1+c-kit+ (LSK) hematopoietic progenitors with the Notch ligand Delta1ext−IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of LSK cells cultured with Delta1ext−IgG compared to recipients of non-cultured LSK cells. Furthermore, data suggest that accelerated T cell recovery resulted from progenitor cells induced towards T cell differentiation. 103 LSK cells cultured with Delta1ext−IgG generated an 108-fold increase in total number of cells compared to cutlure with control IgG. Using limiting dilution analysis we estimated the number of progenitors with T cell potential in 103 LSK cells was 2.3; after culture with Delta1ext−IgG, the number in the 1011 expanded product was 1.9×109, representing a 5×108-fold increase. Lethally irradiated mice received 105 BM cells along with 103 LSK cells or 106 Delta1ext−IgG-cultured cells. At 3 and 5 wks after HCT, blood samples from recipients of Delta1ext−IgG-cultured cells contained 9- and 3-fold higher numbers of CD3+ cells compared to recipients of LSK cells (p